Galectin-receptor interaction: a key player in liver fibrosis induced by Schistosoma japonicum infection

Background Schistosoma japonicum eggs lodge in the liver and induce a fibrotic granulomatous immune response in the liver of host. Galectin 3 (Gal-3) is a protein implicated in fibrosis in multiple organs. However, the pathology and molecular mechanisms promoting hepatic granuloma formation remain poorly understood. Methods To investigate the effect of blocking galectin-receptor interactions by α-lactose on liver immunopathology in mice with S. japonicum infection, C57BL/6 mice were infected with S. japonicum and alpha (α)-lactose was intraperitoneally injected to block the interactions of galectins and their receptors. Results Compared with S. japonicum-infected mice, there were significantly decreased Gal-3 mRNA and protein expression levels, decreased intensity of Gal-3 fluorescence in the liver, decreased serum ALT and AST levels, decreased egg numbers of S. japonicum in the liver section, attenuated hepatic and spleen pathology, and alleviated liver fibrosis accompanied with decreased protein expression levels of fibrosis markers [α-smooth muscle actin (α-SMA), collagen I, and collagen IV] in the liver of S. japonicum-infected mice blocked galectin-receptor interactions with hematoxylin-eosin staining, Masson’s trichrome staining, immunohistochemistry, or Western blot analysis. Compared with S. japonicum-infected mice, blocking galectin-receptor interactions led to increased eosinophil infiltration and higher eosinophil cationic protein (ECP) expression in the liver, accompanied by increased mRNA levels of eosinophil granule proteins [ECP and eosinophil peroxidase (EPO)], IL-5, CCL11, and CCR3 in the liver and decreased mRNA levels of Gal-3 and M2 macrophage cytokines (TGF-β, IL-10, and IL-4) in the liver and spleen by using quantitative real-time reverse transcription-polymerase chain reaction. In addition, there were increased Beclin1 protein expression and protein expression ratio of LC3B-II/LC3B-I and decreased p62 protein expression and protein expression ratios of phospho-mTOR/mTOR and phospho-AKT/AKT by Western blot; increased double-labeled F4/80+/LC3B+ cells by immunofluorescence staining; increased M1 macrophage polarization in the liver of S. japonicum-infected mice blocked galectin-receptor interactions by flow cytometric analysis and immunofluorescence staining. Conclusions Our data found that blockage of galectin-receptor interactions downregulated Gal-3, which in turn led to reduced liver functional damage, elevated liver eosinophil recruitment, promoted macrophage autophagy through the Akt/mTOR signaling pathway, and alleviated liver pathology and fibrosis. Therefore, Gal-3 plays a pivotal role during S. japonicum infection and could be a target of pharmacologic potential for liver fibrosis induced by S. japonicum infection. Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1186/s13071-024-06314-5.


Background
Schistosomiasis is one of the serious tropical diseases caused by three main species of schistosomes: Schistosoma mansoni, S. haematobium, and S. japonicum [1].According to the World Health Organization report, at least 251.4 million people required preventive treatment for schistosomiasis in 2021, out of which > 75.3 million people were reported to have been treated [2].Schistosoma japonicum infection causes granulomatous responses to parasite eggs trapped in the liver, resulting in severe liver fibrosis and irreversible impairment of the liver and even death of the host [3].So far, the mechanism of egg-induced liver fibrosis in S. japonicum infection remains unclear.Therefore, exploring the aspects of immunology and interplay between host and parasites during S. japonicum infection may have essential roles in schistosomiasis japonica-induced liver fibrosis.
Galectins are a family of carbohydrate-binding proteins that are expressed by a wide variety of cells and bind to galactose-containing glycans; they are involved in many physiological functions such as inflammation, immune responses, cell migration, autophagy, and signaling, and 15 members have been identified in various cells and tissues [4].Galectin (Gal)-1 and Gal-3 facilitate the proliferation of hepatic stellate cells and play an important role in liver fibrosis [5], in which Gal-1 is a promising molecular target for the development of therapeutic tools to treat fibrosis in different chronic diseases [6].Gal-3 is a chimeric lectin and potent driver of many aspects of fibrosis, and Gal-3 binds to Schistosoma egg antigens and promotes liver fibrosis of S. mansoni-infected C57BL/6 mice [7].Our previous study found that Gal-1, Gal-3, eosinophils, and macrophages may participate in the development of egg granulomatous inflammation and fibrosis induced by S. japonicum infection [8]; however, the mechanisms remain unknown.
Macrophage autophagy has a critical regulatory function in downregulation of liver immunopathology in different kinds of diseases.It has been reported that S. japonicum egg antigen-triggered macrophage autophagy limited the development of pathology in the liver of C57BL/6 mice [9].Galectins expressed by immune cells can participate in host responses to infection by directly binding to microorganisms or by modulating antimicrobial functions such as autophagy [10].However, the role of galectins in regulating liver fibrosis caused by S. japonicum infection remains poorly understood, and little is known about whether and how galectins can impact macrophage autophagy and the liver immunopathology after schistosome infection.Elucidating the molecular mechanisms that promote or restrict tissue fibrosis may lead to more effective strategies for immunological intervention in schistosomiasis.By blocking galectinreceptor interactions with α-lactose, our data demonstrated that Gal-3, eosinophils, macrophages, and autophagy may play important roles in liver pathology and fibrosis caused by S. japonicum infection.Therefore, Gal-3 could be a target for early therapy of hepatic fibrosis induced by S. japonicum infection.

Ethics statement
All the experimental animals were housed in groups of five and given 5 days to acclimate to the housing facility.The mice were housed in an air-conditioned room (22-25 °C), maintained under a 12-h light-dark cycle, and water and food were supplied ad libitum.The health of mice was monitored twice daily, and no adverse events were observed during feeding.All surgeries were performed under anesthesia using isoflurane, and the appropriate organs were harvested when mice were killed.

Schistosoma japonicum and experimental infection
Female, 6-8-week-old C57BL/6 mice were obtained from the Animal Center of Sun Yat-sen University (Guangzhou, China).Oncomelania hupensis snails were purchased from the National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention (Shanghai, China).A total of 40 C57BL/6 mice were used in this study, which were randomly assigned to 4 groups of 10 in each group: (i) naive group: mice were intraperitoneal (i.p.) injected with PBS daily as uninfected control; (ii) α-lactose-treated group (lact group): mice were i.p. injected with α-lactose (L8040-100, Sigma-Aldrich, St. Louis, MO, USA) solution in PBS alone as α-lactose control; (iii) S. japonicum-infected group (Sj group): mice were percutaneously infected with 30 cercariae of S. japonicum, and (iv) S. japonicum + α-lactose group (Sj + lact group): mice were percutaneously infected with 30 cercariae of S. japonicum and i.p. injected with α-lactose treatment.Because male-female adult parasites pairing is common at 18 days after S. japonicum infection in mouse models [11], lact group and Sj + lact group were administered 1.5 mM of α-lactose PBS solution daily from 19 days post infection (p.i.) to 8 weeks p.i., while naive group was i.p. injected with equal volume of PBS daily.At week 8 post-S.japonicum infection, each group of mice was i.p. anesthetized with isoflurane and weighed, blood samples were drawn from the orbit to determine serum levels of aspartic aminotransferase (AST) and alanine aminotransferase (ALT), and livers and spleens were harvested, weighed, and then stored based on different experimental needs.

Measurement of serum enzyme activities
The whole blood samples of mice were collected and kept at room temperature for 2 h and then centrifuged at 3000 rpm for 10 min after coagulation.The supernatant was harvested, and the serum levels of AST and ALT were measured to evaluate liver function on an automatic biochemical analyzer (Chemray 800; Rayto Life and Analytical Sciences Co., Shenzhen, China) by different assay kits (Rayto Life and Analytical Sciences Co.) according to the manufacturer's instructions.

Determination of liver and spleen indexes
The body weight of each mouse in different groups was measured before they were killed, and the livers and spleens were excised and weighed.Liver index = liver weight (g)/body weight (g) × 100%, and spleen index = spleen weight (g)/body weight (g) × 100%.

Histopathological examination
The liver and spleen of mice were collected, and part of the tissues was formaldehyde-fixed in 10% buffered natural formaldehyde (Guangzhou Chemical Reagent Factory, Guangzhou, China) for over 48 h, paraffin-embedded, and sectioned.Tissue sections (4 µm) were stained with hematoxylin-eosin (H&E) or Masson's trichrome solution (Wuhan Servicebio Technology Co., Ltd., Wuhan, China).A semi-quantitative scoring system was used to evaluate histological changes in liver tissues from four mice of each group.The histopathological changes were determined under 200 × magnification in five noncontiguous sections.Microscopic scores of the inflammation severity were graded into four grades: 0, no inflammation; 1, mild inflammation; 2, moderate inflammation and necrosis, 3, severe inflammation and necrosis [12].The eosinophil numbers in liver tissues were quantified using images taken with a digital camera system under 1000 × magnification measured using Image Pro plus 6.0 software (Media Cybernetics, Inc., MD, USA).The egg numbers of S. japonicum in liver sections were counted in images taken with a digital camera system under 200 × magnification.Eosinophil density was expressed as the number of eosinophils per square millimeter in the liver.Collagen appears blue using Massonʼs trichrome staining, and the blue area reflects the amount of collagen.The percentage of the area stained with blue color in total area was estimated quantitatively by using Image Pro Plus 6.0 software (Media Cybernetics, Inc.) under 100 × magnification [13], and data are expressed in units of area.The severity of fibrosis was distinguished by the area of collagen.

Flow cytometry
Liver tissue from each mouse was cut into small pieces, and liver leukocytes were separated by Ficoll-Hypaque (Haoyang Biological Manufacture Co., Ltd., Tianjin, China) gradient centrifugation after being filtered through a 70-µm cell sieve.Spleen tissue from each mouse was also rendered into single-cell suspensions by dispersing through a 70-µm cell sieve.The cell suspension was purified with ACK lysis buffer, and cell concentration was adjusted appropriately.For flow cytometry analysis, liver leukocytes and spleen lymphocytes were washed twice and suspended in pre-chilled PBS with 0.1% BSA.A total of 1 × 10 6 cells/100 µl were incubated with PE/Cyanine7-conjugated anti-CD45 antibody, EV450-conjugated anti-F4/80 antibody, APC-conjugated anti-CD11b antibody, PE-conjugated anti-MHC-II antibody, and Ghost Dye ™ Red 780 (ELGBIO Biotechnology Co., Ltd., Guangzhou, China) for surface staining, and then the cells were re-suspended with fixation/permeabilization buffer (BD Biosciences Inc., New York, USA) and incubated with FITC-conjugated anti-CD206 antibody (ELGBIO Biotechnology Co., Ltd.) for intracellular staining.Data were detected with a CytoFLEX flow cytometer and analyzed using FlowJo software (Treestar Inc., CA, USA).

Determination of mRNA expression by using quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR)
Total RNA was isolated from liver and spleen tissues using FastPure ® Cell/Tissue Total RNA Isolation Kit (Vazyme Biotech Co., Nanjing, China) according to the manufacturer's instructions.Then, cDNA was synthesized from total RNA using PrimeScript ™ II 1st Strand cDNA Synthesis Kit (TaKaRa Bio Inc., Shiga, Japan).To determine the mRNA levels of Gal-1, Gal-3, Gal-9, eosinophil cationic granule proteins [ECP, eosinophil peroxidase (EPO)], and eosinophil chemokines [IL-5, C-C motif chemokine 11 (CCL11), and CC chemokine recep-tor3 (CCR3)] in the liver tissue, and the mRNA levels of M1 macrophage-associated genes (iNOS and IL-1β) and M2 macrophage-associated genes (TGF-β, Arg1, IL-4, and IL-10) in the liver and spleen tissues, qRT-PCR was performed in a 10 µl volume with the SYBR Green qPCR Master Mix (TaKaRa Bio Inc.).The PCR was run on a CFX96 Touch ® Real-Time PCR Detection System (Bio-Rad Laboratories, CA, USA) for 30 s at 95 °C, followed by 39 cycles of 5 s at 95 °C and 30 s at 60 °C.Then, the melting curve was analyzed (95 °C for 15 s and 65 °C for 15 s).Relative gene expression levels were determined using the 2 −ΔΔCt method with Light Cycler 480 software (Roche, version 1.5.0).Mouse housekeeping gene, GAPDH, was used as the control to normalize specific mRNA expression levels.The fold change in the expression of each target gene mRNA relative to GAPDH in each group was determined using the 2. −ΔΔCt method.All primers are listed in Table 1.

Statistical analysis
All data are presented as the mean ± standard deviation (SD) and were analyzed using SPSS 20.0 software to test normality.SPSS 20.0 software was used to conduct t-test of independent samples (for comparison between two groups of data) or one-way ANOVA (for comparison between multiple groups of data) for statistical analysis.All histograms were designed with GraphPad Prism 8 (GraphPad software, San Diego, CA, USA).P < 0.05 was considered statistically significant.

Blockage of galectin-receptor interactions decreased Gal-3 level in the liver of S. japonicum-infected mice
Only a few Gal-1, Gal-3, and Gal-9 positive cells labeled dark brown were observed in the liver section of naive group and lact group.However, there were many Gal-1, Gal-3, and Gal-9 positive cells in the liver section of Sj group (Fig. 1A).Compared with uninfected controls, there were significantly increased Gal-1, Gal-3, and Gal-9 positive cells in the liver of Sj group (P < 0.0001).Compared with Sj group, there were significantly decreased Gal-3 positive cells in the liver of Sj + lact group (P < 0.001); however, the numbers of Gal-1 or Gal-9 positive cells had no statistical significance (P > 0.05, Fig. 1B) (ANOVA; F Gal-9 = 173.667,F Gal-3 = 239.084,F Gal- 1 = 53.449;P < 0.0001).Consistent results were also found by Western blot; only Gal-3 protein expression was significantly decreased in the liver of Sj + lact group compared to Sj group (P < 0.05, Fig. 1C, 1D).

Blockage of galectin-receptor interactions relieved liver function damage of S. japonicum-infected mice
Gross observation of liver specimens found that the livers of normal control mice (either naive mouse or α-lactosetreated mouse) were light red color with a smooth surface; in contrast, the livers of S. japonicum-infected mice (either Sj mouse or Sj + lact mouse) were dark red color with numerous irregular whitish spots on the surface, and the spleens were markedly enlarged and darkened (Fig. 2A).
The liver and spleen indexes of Sj group and Sj + lact group were significantly higher than those of naive group (P < 0.0001 and P < 0.001, respectively) and lact group (P < 0.0001 and P < 0.001, respectively).Compared with Sj group, the liver and spleen indexes of Sj + lact group were significantly increased (P < 0.05) (ANOVA; F liver indexes = 92.410,F spleen indexes = 50.484;P < 0.0001, Fig. 2B, C).The results suggested that blockage of galectin-receptor interaction was beneficial to alleviate liver and spleen damage induced by chronic S. japonicum infection.Serum levels of ALT and AST are used to assess liver function.Compared with naive group and lact group, serum levels of ALT and AST were significantly increased in Sj group (P < 0.01) and Sj + lact group (P < 0.05) (ANOVA; F ALT = 41.679,F AST = 23.687;P < 0.0001).Compared with Sj group, serum ALT and AST levels were significantly decreased in Sj + lact group (P < 0.01 and P < 0.05, respectively, Fig. 2D, E).

Blockage of galectin-receptor interactions promoted eosinophil infiltration in the liver and attenuated the pathology of liver and spleen of S. japonicum-infected mice
The livers and spleens of different groups of mice were examined histologically at 8 weeks p.i. Compared with uninfected controls, severe inflammation and significantly increased eosinophil infiltration around egg granulomas were observed in the liver sections of Sj group and Sj + lact group (Fig. 3A).The numbers of eosinophils in liver sections of different groups were counted under 1000 × magnification.Quantitative analysis showed that compared with naive group or lact group, the numbers of eosinophils were significantly increased in the livers of Sj group (P < 0.001) and Sj + lact group (P < 0.001), and eosinophil number was significantly increased in the liver of Sj + lact group compared with Sj group (P < 0.01, Fig. 3B) (ANOVA; F ALT = 41.679,F AST = 23.687;P < 0.0001).
The mRNA expression levels of eosinophil granule proteins (ECP and EPO) and eosinophil chemokines (IL-5, CCL11, and CCR3) in the liver were determined.Compared with naive group or lact group, the mRNA levels of ECP (P < 0.0001), EPO (P < 0.001 and P < 0.0001, Histological observation showed that no pathological change was observed in the liver and spleen tissues of naive group and lact group.However, severe histological changes were observed in the liver and spleen tissues, and diffuse S. japonicum egg granulomas were surrounded by many inflammatory cells in the liver and spleen tissues of Sj group, while attenuated granulomatous inflammation surrounding egg granulomas was observed in the liver (Fig. 3A) and spleen (Fig. 3F) tissues of Sj + lact group.Furthermore, the egg numbers of S. japonicum in the liver sections of Sj + lact group was lower than in Sj group (t-test, t liver = 2.367, P < 0.05, Fig. 3D).Compared with Sj group, semi-quantitative histopathological scores based on histopathological changes in the liver (Fig. 3E) and spleen (Fig. 3G) tissues were significantly decreased in Sj + lact group (t-test, t liver = 3.468, P < 0.01 and t spleen = 2.483, P < 0.05, respectively).Therefore, our data demonstrated a profound protective role of eosinophils against S. japonicum; however, the molecular mechanism had not been elucidated.
Then, we used double-immunofluorescence staining to verify whether eosinophil infiltration was regulated by Gal-3 in the liver of S. japonicum-infected mice.It has been reported that Gal-3 is predominantly located in the cytoplasm, but it also shuttles into the nucleus and is secreted onto the cell surface and into biological fluids [14].ECP is a basic and potentially cytotoxic granule protein stored in an inactive form in the granules; it is released from the eosinophil upon activation [15].Our result showed that a faint intensity of Gal-3 fluorescence (Gal-3 + , green) and ECP fluorescence (ECP + , eosinophils marker, red) was observed in the liver of naive group and lact group.Compared with uninfected controls, a higher intensity of Gal-3 fluorescence and ECP fluorescence was observed in the liver sections of Sj group and Sj + lact group.Compared with Sj group, a lower intensity of Gal-3 fluorescence and a higher intensity of ECP fluorescence were observed in the liver section of Sj + lact group (Fig. 4).Thus, blockage of galectin-receptor interaction may induce eosinophil infiltration through downregulation of Gal-3 expression in the liver of S. japonicuminfected mice.

Blockage of galectin-receptor interactions relieved liver fibrosis of S. japonicum-infected mice
To determine whether blockage of galectin-receptor affects liver fibrogenesis, hepatic collagen deposition was determined by Masson's trichrome staining.There was a large amount of collagen fiber deposition around the granulomas in the liver section of Sj group (Fig. 5A).Compared with Sj group, there was significantly decreased fibrotic area in the liver section of Sj + lact group (P < 0.001, Fig. 5B) (ANOVA; F = 83.851;P < 0.0001).
The observation by immunohistochemical staining revealed that there were only a few and uniform distribution of α-SMA, collagen I, and collagen IV positive cells (stained brown) observed in the liver sections of naive group and lact group (Fig. 6A).However, compared with uninfected controls, there were increased α-SMA, collagen I, and collagen IV positive areas around egg granulomas in the liver sections of Sj group (P < 0.0001, P < 0.001, and P < 0.001, respectively) and Sj + lact group (P < 0.001, P < 0.01, and P < 0.001, respectively).Compared with Sj group, there were

Blockage of galectin-receptor interactions promoted M1 macrophage polarization in the liver and spleen of S. japonicum-infected mice
The expressions of MHC-II (a phenotypic marker for M1 macrophages) and CD206 (a phenotypic marker for M2 macrophages) in the liver of different groups of mice were compared using immunofluorescence staining, and macrophages were detected by staining with anti-F4/80 antibody.A few F4/80 positive cells (F4/80 + cells, red fluorescence) were observed in the liver sections of naive group and lact group, but many F4/80 + cells were observed in the liver sections of Sj group and Sj + lact group (Fig. 7A, C).Meanwhile, compared with naive group and lact group, more MHC-II positive cells (MHC-II + cells, green fluorescence) or CD206 positive cells (CD206 + cells, green fluorescence) and merged with F4/80 + cells in yellow color were observed in the liver sections of Sj group and Sj + lact group (Fig. 7A, C).The numbers of MHC-II + cells and CD206 + cells in the liver tissues of Sj group and Sj + lact group were significantly higher than those of naive group and lact group (ANOVA; F MHC-II = 462.657,F CD206 = 229.691;P < 0.0001, Fig. 7B, D).Compared with Sj group, the number of MHC-II + cells was significantly increased (P < 0.0001), whereas the number of CD206 + cells was significantly decreased (P < 0.0001) in the liver tissue of Sj + lact group.
The gating strategy of flow cytometry data used in the liver samples of Sj + lact group was as below; this was also used in all liver and spleen samples of different groups: (i) gated the leukocyte subsets from all cells according to the parameters of SSC-A/FSC-A, namely P1; (ii) gated the single cells from P1 according to FSC-A/FSC-A parameters, namely P2; (iii) gated the live cells from P2 according to SSC-A/LIVE/DEAD parameters, namely P3; (iv) F4/80 + /CD11b + cells (Q1-UR, top right corner) were gated from the cells of the P3, which were macrophage subtypes (Fig. 8A).Based on the above gating data, the M1/M2 macrophage subtypes of antibody-labeled MHC-II and CD206 cells in the livers and spleens of mice of different groups were further analyzed (Fig. 8B).Compared with naive group and lact group, there were significantly increased CD206 cells in the liver and spleen of Sj group (P < 0.001); however, the proportion of MHC-II cells had no significant change.Compared with Sj group, there were significantly increased MHC-II cells in the liver (P < 0.001) and spleen (P < 0.001) and significantly decreased CD206 cells in the liver (P < 0.01) of Sj + lact group (ANOVA; F liver MHC-II = 42.127,F liver CD206 = 13.868,F spleen MHC-II = 25.253,F spleen CD206 = 16.672;P < 0.0001, Fig. 8C).These findings suggest that S. japonicum infection induced increased M2 macrophages, while blockage of galectin-receptor interactions resulted in increased M1 macrophages and decreased M2 macrophages in the liver of Sj + lact mice.

Blockage of galectin-receptor interaction promoted macrophage autophagy through the Akt/mTOR signaling pathway in the liver of S. japonicum-infected mice
Study has reported that Gal-3, as a negative regulator of autophagy, can decrease autophagy activity [16].To verify whether autophagy is regulated by Gal-3 in the liver of S. japonicum-infected mice, immunofluorescence staining showed that faint intensities of LC3B fluorescence and Gal-3 fluorescence were observed in the livers of naive group and lact group.Compared with uninfected mice, there were higher intensities of LC3B fluorescence and Gal-3 fluorescence in the liver of Sj group.Compared with Sj group, the intensity of LC3B fluorescence was stronger and the intensity of Gal-3 fluorescence was lower in the liver of Sj + lact group (Fig. 11A).Thus, blockage of galectin-receptor interaction may promote autophagy through downregulating Gal-3 expression in the liver of S. japonicum-infected mice.
According to the above results, we found that blockage of galectin-receptor interaction could promote autophagy in the liver of Sj + lact group; however, which kind of cells undergoes autophagy needs to be investigated.By immunofluorescence staining, a few F4/80 positive cells (F4/80 + cells with red fluorescence) were observed in the liver sections of naive group and lact group, but many F4/80 + cells were observed in the liver sections of Sj group and Sj + lact group (Fig. 12A).Compared with naive group and lact group, more doublelabeled cells (F4/80 + /LC3B + cells) merged in yellow color were counted in the liver section of Sj group (P < 0.001).Compared with Sj group, the number of F4/80 + /LC3B + cells was significantly increased (P < 0.001) in the liver section of Sj + lact group (ANOVA; F = 153.973;P < 0.001, Fig. 12B).The results suggest that macrophage autophagy may be involved in regulating liver pathogenesis of S. japonicum-infected mice.

Discussion
Schistosomiasis is a parasitic disease caused by trematode blood flukes of the genus Schistosoma.It affects almost 240 million people worldwide, and > 700 million people live in endemic areas [2].The formation of granulomata around eggs of S. japonicum deposited in the liver is related to hepatic fibrogenesis in schistosomiasis japonica [17].However, the mechanism of hepatic fibrosis caused by S. japonicum egg granuloma remains not well defined.
Galectins are lectins with a highly conserved carbohydrate recognition domain (CRD) and affinity for β galactose-containing oligosaccharides [18].Gal-3 is predominantly located in the cytoplasm with multiple functions including regulation of inflammation, cell growth, signaling, chemotaxis, and cell-matrix interactions, etc. [19].Gal-3 has been described as a biomarker associated with fibrosis and inflammation in patients with heart failure [20].In the present study, serum ALT and AST levels, the liver and spleen indexes, liver and spleen histopathological score, the grade of hepatic fibrosis, and Gal-3 expression level in the liver were significantly decreased in Sj + lact group compared with Sj group.It has been reported that Gal-3 is involved in Chagas disease and mediating cardiac tissue damage as well as in immune responses against Trypanosoma cruzi experimental infection; lack of Gal-3 prevents cardiac fibrosis [21].Genetic deletion of Gal-3 prevented cardiac damage, adverse remodeling, and dysfunction, associated with reduced cardiac oxidative stress and fibrosis in C57 Gal-3 knockout mice treated with doxorubicin [22].The role of Gal-3 in fibrosis and immune response has been extensively studied.In the present study, our results found that Gal-3 was strongly involved in hepatic immunopathology induced by S. japonicum infection, and blockage of galectin-receptor interactions resulted in relieved liver function and decreased liver pathology and fibrosis in S. japonicum-infected C57BL/6 mice, indicating the potential therapeutic effect of Gal-3 in fibrotic pathogenesis caused by S. japonicum infection.
It has been reported that the eggs of S. japonicum and S. mansoni induced local granulomas inflammatory response, and granulomas mainly consist of lymphocytes, macrophages, and eosinophils [17].The role of eosinophils in the pathogenesis of schistosomiasis remains controversial [23].Study has demonstrated that eosinophils play a role in limiting the pathology associated with human schistosomiasis mansoni [24], and ECP may be an important protein both in the immune response against S. mansoni and in the development of periportal fibrosis [24].However, experiments using S. mansoni-infected mouse models of eosinophil lineage ablation demonstrated that eosinophils had no impact on granuloma number, size, or fibrosis, and eosinophil ablation also had no effect on worm burden or egg deposition [25].Therefore, eosinophils may make unknown immunomodulatory contributions to liver fibrosis of mice infected with S. mansoni [25].In the absence of Gal-3, S. mansoni egg granulomas in chronic-phase are smaller in Fig. 11 Effects of α-lactose on autophagy Akt/mTOR signaling pathway in the livers of different groups of mice.A Liver sections of different groups of mice were co-stained with anti-Gal-3 (red) and anti-LC3B (autophagy marker, green).DAPI was used to visualize nuclei (blue).Original magnification 1000 × (scale bar = 10 µm).B Western blot analysis for p-mTOR, mTOR, p-AKT, AKT, p62, Beclin1, LC3B, and GAPDH.C p-mTOR, p-AKT, and LC3B-II band density normalized to mTOR, AKT, and LC3B-I, respectively; the relative densitometry of p62 and Beclin1 (relative to the endogenous control, GAPDH) expressed as fold change.The values are expressed as mean ± SD (n = 4).ns: no significance; # P < 0.05 and ## P < 0.01, Sj group compared with naive group and lact group; * P < 0.05 and ** P < 0.01, Sj + lact group compared with Sj group (See figure on next page.)diameter, with thinner collagen fibers [26].Schistosoma mansoni-infected Gal-3 -/-mice have fewer macrophages and B lymphocytes but increased eosinophil absolute number in the periphery compared with the infected wild-type mice [26].The inflammatory cell composition around S. mansoni egg granulomas in Lgals3 -/-mice presents dominance of eosinophils in liver granulomas, and there is significant inflammatory infiltration as well as phagocytes (specifically Kupffer cells) and numerically reduced and diffuse matrix extracellular deposition in fibrotic areas in the liver of Lgals3 −/− infected mice compared to Lgals3 +/+ mice [7].In the present study, there were decreased Gal-3 expression, increased eosinophil infiltration and ECP expression, and increased mRNA levels of ECP, EPO, IL-5, CCL11, and CCR3 in the liver of Sj + lact group compared to Sj group.However, hepatic histopathological changes and fibrosis were significantly alleviated, accompanied by significantly decreased α-SMA, collagen I, and collagen IV in the liver of Sj + lact group.Our data further demonstrated that blockage of galectin-receptor interactions increases eosinophil numbers and eosinophil degranulation in the liver of S. japonicum-infected mice, which has a great impact on the immunomodulation of granulomatous inflammation and decreases hepatic lesions induced by egg deposition of S. japonicum.
Gal-3 is an immune regulator and induces the immune response against S. mansoni infection, and the absence of Gal-3 interfered with the function of the resident liver macrophages and Kupffer cells [7].Because Gal-3 is highly expressed in inflammatory cells surrounding liver granulomas, it is considered to favor the interactive network between macrophages and immunogenic carbohydrates [27].Gal-3 is highly expressed by Kupffer cells in liver around S. mansoni eggs [7].Compared with S. mansoni-infected Lgals3 +/+ mice, there was significant inflammatory infiltration with lower levels of α-SMA and eotaxin and higher levels of IL-4 in S. mansoni-infected Lgals3 -/-mice at 90 days p.i.The reduction of macrophages and decreased phagocytic activity of local Kupffer cells in S. mansoni-infected Lgals3 -/-mice suggest that Gal-3 is necessary for fibrotic Fig. 12 Double immunofluorescence staining of F4/80 and LC3B in the liver tissues of different groups of mice.A Liver sections of different groups of mice were co-stained with anti-F4/80 (macrophage marker, red) and anti-LC3B (autophagy marker, green).DAPI was used to visualize nuclei (blue).Original magnification 1000 × (scale bar = 10 µm).B F4/80 + /LC3B + cells in the liver tissue sections counted in five random fields were quantified.Data are shown as the means ± SD (n = 4).### P < 0.001, Sj group compared with naive group and lact group; *** P < 0.001, Sj + lact group compared with Sj group concentric granulomas and myofibroblast activation in S. mansoni-infected mice [7].In the present study, blockage of galectin-receptor interactions resulted in decreased M2 macrophages and increased M1 macrophages in the liver of Sj + lact mice.It has been reported that iNOS and IL-1β are M1 macrophage-associated genes, and TGF-β, Arg1, IL-4, and IL-10 are M2 macrophage-associated genes [28].We evaluated M1 and M2 macrophage cytokines in the liver and spleen of mice of different groups; there were significantly increased levels of iNOS and IL-1β in the liver and spleen, and significantly decreased levels of TGF-β, IL-10, Arg-1, and IL-4 in the liver and significantly decreased levels of TGF-β, IL-10, and IL-4 in the spleen of Sj + lact group compared to Sj group.Additionally, our results showed that there were significant negative correlations between the mRNA levels of Gal-3 and M1 macrophage cytokines (iNOS/IL-1β) and significant positive correlations between the mRNA levels of Gal-3 and M2 macrophage cytokines (TGF-β/ Arg-1/IL-10/IL-4) in the liver of Sj + lact group.Our data demonstrated that blockage of galectin-receptor interactions increases M1 macrophage response and decreases M2 macrophage response in the liver and spleen of S. japonicum-infected mice, indicating that Gal-3 is associated with macrophage polarization.
Macrophage autophagy has an important regulatory role in downregulation of hepatic immunopathology in different diseases, and macrophages play a critical role in S. japonicum egg-triggered immunopathology in host liver [9].Alveolar macrophages are significant sources of Gal-3, which drives inflammation and the development of pulmonary fibrosis and reduction in Gal-3 expression in alveolar macrophages and neutrophils decreasing inflammation and neutrophil recruitment into the interstitium of lung [29].In the present study, by immunofluorescence staining, more F4/80 + /LC3B + cells were observed in the liver of Sj + lact group compared to Sj group.It has been reported that Gal-3, as a negative regulator of autophagy, can decrease autophagy activity [16].Our result is consistent with this study.
A study has reported that the PI3K/Akt/mTOR signal has a critical role in the biology of S. mansoni; it is involved in glucose uptake by S. mansoni, the reproduction of S. mansoni, and worm survival [30].However, whether the Akt/mTOR pathway can impact on liver immunopathology caused by S. japonicum infection through regulating macrophage autophagy has not been reported.Enterococcus faecalis lipoteichoic acid may increase macrophage autophagy by inhibiting the PI3K/Akt/mTOR pathway [31].Doxazosin inhibits autophagy by activation of the PI3K/Akt/mTOR signaling pathway in hepatic stellate cells [32].In the present study, our results showed that Gal-3 expression was decreased while LC3B expression was increased in the liver of Sj + lact group compared to Sj group.There were decreased p62 protein expression and decreased protein expression ratios of phospho-mTOR/ mTOR and phospho-AKT/AKT, while increased Bec-lin1 protein expression and increased protein expression ratio of LC3B-II/LC3B-I in the liver of Sj + lact group at 8 weeks p.i. compared with Sj group, indicating that blockage of galectin-receptor interaction (e.g. through downregulating Gal-3 expression) may reduce S. japonicum-induced liver fibrosis by promoting autophagy by inhibiting AKT/mTOR signaling pathway activation.Gal-3 knockdown may enhance autophagy by inhibiting the Gal-3/Akt/mTOR pathway, which helps ameliorate renal fibrosis [33].We proposed that autophagy signaling pathway could be involved in the pathogenesis of schistosomiasis japonica.Further study focused on macrophage autophagy could elucidate the pathological mechanisms of liver fibrosis caused by S. japonicum infection.
In conclusion, we used mouse models to investigate whether galectins were involved in the immunopathological mechanisms of liver fibrosis caused by S. japonicum infection by blocking galectin-receptor interactions with α-lactose; our data suggested that Gal-3 plays a pivotal role during S. japonicum infection, which leads to attenuated liver functional damage and liver fibrosis by promoting eosinophil infiltration, M1 macrophage polarization, and macrophage autophagy.Thus, use of Gal-3 inhibition may be a therapeutic approach to prevent the early liver fibrosis caused by S. japonicum infection.

Fig. 2
Fig. 2 Blockage of galectin-receptor interactions alleviated the liver function damage of S. japonicum-infected mice.A Gross specimens of liver and spleen of different groups of mice.B The liver indexes of different groups of mice.C The spleen indexes of different groups of mice.D Serum ALT levels of different groups of mice.E Serum AST levels of different groups of mice.Results are expressed as mean ± SD (n = 7).## P < 0.01, ### P < 0.001, #### P < 0.0001, Sj group compared with naive group and lact group; * P < 0.05, ** P < 0.01, Sj + lact group compared with Sj group

Fig. 3
Fig. 3 The liver and spleen histopathology of different groups of mice.Mice were infected with 30 Schistosoma japonicum cercariae and killed at 8 weeks p.i.A Representative microscopic pictures of the liver sections of different groups stained with H&E.No histological change was observed in the liver and spleen of naive group and lact group; egg granulomas and a large number of eosinophils around S. japonicum egg granulomas were observed in the liver and spleen of Sj group and Sj + lact group.Original magnifications of the images are 400 × (scale bar = 20 µm) and 1000 × (scale bar = 9 µm).B Eosinophil count analysis in the liver tissues.Data are represented as mean ± SD.There were six mice per group.C The mRNA expressions of ECP, EPO, IL-5, CCL11, and CCR3 in the livers of different groups of mice at 8 weeks p.i. Values are means from triplicate measurements, and data are presented as means ± SD (n = 10).D The egg numbers of S. japonicum in the livers.Data are represented as mean ± SD (n = 5).E Histopathological score analysis of the liver tissues.F Representative microscopic pictures of the spleen sections of different groups of mice stained with H&E.G Histopathological score analysis of the spleen tissues.Original magnification 200 × (scale bar = 100 µm).Data are represented as mean ± SD (n = 4).### P < 0.001 and #### P < 0.0001, Sj group compared with naive group and lact group; * P < 0.05 and ** P < 0.01, Sj + lact group compared with Sj group

Fig. 4
Fig. 4 Double immunofluorescence staining of Gal-3 and ECP in the liver tissues of different groups of mice.Fluorescence microscopy observation of liver tissues in different groups of mice stained with anti-Gal-3 (green) and anti-ECP (eosinophils marker, red).Tissues were counterstained with DAPI (blue).Original magnification 1000 × (scale bar = 10 µm)

Fig. 5
Fig. 5 Hepatic fibrosis was relieved in Schistosoma japonicum-infected mice with blockage of galectin-receptor.A Representative liver granulomas were observed by Massonʼs trichrome staining at 8 weeks p.i. after S. japonicum infection.Collagen is in blue color.Original magnification 100 × (scale bar = 200 µm).B Collagen deposition positive areas were digitized and analyzed on Image Pro Plus software.Results are expressed as mean ± SD (n = 6).#### P < 0.0001, Sj group compared with naive group and lact group; *** P < 0.001, Sj + lact group compared with Sj group

Fig. 6 Fig. 7
Fig. 6 Immunohistochemical staining of α-SMA, collagen I, and collagen IV in the liver.A Immunohistochemical staining of α-SMA, collagen I, and collagen IV positive cells in the livers of different groups of mice.Original magnification 400 × (scale bar = 50 µm).B α-SMA, collagen I, and collagen IV positive areas were digitized and analyzed by Image Pro Plus software.Data are expressed as mean ± SD (n = 4).C The protein expression levels of α-SMA, collagen I, and collagen IV in the liver were detected by Western blot.D Densitometric analysis of α-SMA, collagen I, and collagen IV normalized to GAPDH and expressed as fold change.Data are expressed as mean ± SD (n = 4).## P < 0.01, ### P < 0.001, #### P < 0.0001, Sj group compared with naive group and lact group; * P < 0.05 and ** P < 0.01, Sj + lact group compared with Sj group

Fig. 10
Fig. 10 Correlation analysis between the mRNA expression levels detected in the livers of different groups of mice.Correlation analysis between the mRNA expression levels of Gal-3 and iNOS/IL-1β/TGF-β/IL-10/Arg-1/IL-4 in the livers of Sj group A and Sj + lact group B (n = 8).The r value generates the theoretical line of best fit, and the P value indicates the significance of the correlation